Ral proteins constitute a family of small GTPases that can be activated by Ras in cells. In the GTP-bound state, Ral proteins bind to RalBP1, a GTPase-activating protein for CDC42 and Rac GTPases. We have used the two-hybrid system in yeast to clone a cDNA for a novel approximately 85-kDa protein that can bind to an additional site on RalBP1. This newly identified protein contains an Eps homology (EH) domain, which was first detected in the epidermal growth factor (EGF) receptor substrate Eps15. Recently, the EH domain of Eps15 has been shown to bind to proteins containing an asparagine-proline-phenylalanine motif. Moreover, EH domains have been found in proteins involved in endocytosis and/or actin cytoskeleton regulation. The RalBP1 associated Eps-homology domain protein, Reps1, is tyrosine-phosphorylated in response to EGF stimulation of cells. In addition, Reps1 has the capacity to form a complex with the SH3 domains of the adapter proteins Crk and Grb2, which may link Reps1 to an EGF-responsive tyrosine kinase. Thus, Reps1 may coordinate the cellular actions of activated EGF receptors and Ral-GTPases.